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Molecular mechanisms of RET-induced apoptosis

We investigated whether RET-induced cell death was dependent on one of the two classical pathways for apoptosis induction (i.e. the death receptor pathway involving caspase-8 activation or the mitochondria-dependent pathway requiring cytochrome c release and caspase-9 activation) and demonstrated that RET induces cell death through an independent pathway requiring caspase-9 probably by forming a caspase-activating complex. In meantime, we are now interested in the importance of caspase-2 in this caspase activating complex.

The biggest part of the work performed during project period was dedicated to the relevance in vivo of the pro-apoptotic function of RET. As stated in the project, P. Mehlen in collaboration with G. Romeo was initially supposed to generate knock-in mice in which RET would have been mutated in its caspase site, a mutation that blocks RET-induced pro-apoptotic activity. The construct for homologous recombination was prepared using the initial construct used by G. Romeo to generate its C620R knock-in mice and further sequenced for verification.

However, while performing the homologous recombination in ES cells, this part of the project was stopped because P. Mehlen was contacted by Dr. Costantini (Columbia University, USA) that has already generated the mice and observed a very interesting phenotype in homozygous D707N/D707N. Indeed, as expected the number of enteric neurons is strongly increased in the small intestine while the distal colon shows aganglionosis. To further demonstrated that apoptosis is required for the appropriate development of the enteric nervous system, P. Mehlen decided to go for an alternative model in chicken. Indeed, P. Mehlen, in collaboration with N. Le Douarin (Collège de France, France), have electroporated neural crest cells from quail embryos with a dominant negative mutant of RET, -i.e. the intracellular cellular domain of RET mutated in D707N behaves like an inhibitor of RET-induced cell death (see the recent paper from P. Mehlen, Thibert et al., 2003, Science, 301, 84-846). The electroporated quail neural crest cells were then introduced in chicken embryos and the migration of quail cells were then followed in the chicken embryos. P. Mehlen has then observed that a delay in neural crest cells migration occurs in electroporated embryos. This delay in a large number of embryos is associated with a dramatic phenotype the in enteric nervous with either ectopic plexi or a complex absence of neuronal differentiation. P. Mehlen is now performing a few sets of accessory experiments i.e., trying to show that the dominant negative electroporated neural crest cells are more resistant to apoptosis that the mock electroporated when cultured ex-vivo- before trying to publish this approach in back-to-back manuscripts together with the mice approach of Dr. Costantini.

P. Mehlen also initiated an alternative approach to show the in vivo importance of RET-induced cell death. In collaboration with Dr. Arumae in Helsinki, P. Mehlen has taken advantage of the powerful system of primary sympathetic neurons that can be committed to apoptosis via withdrawal of GDNF. Up to now, the overall idea says that this death commitment is related to a loss of survival signals (PI3K, MAPK) normally provided by the canonical GDNF/RET pathways resulting in a by default cell death. However, P. Mehlen took advantage of the dominant negative mutant of RET (see above). Microinjection of the dominant negative mutant was performed in GDNF-withdrawn neurons and cell death was analyzed.

While the injection of this dominant negative mutant has no effect on cell death observed after NGF withdrawal, this mutant strongly inhibited the death of sympathetic neurons withdrawn in GDNF, thus demonstrating that the so-called death by default observed after GDNF loss is related to the pro-apoptotic effect of RET. This is an important observation in regards to the classic cancer therapy strategy that deals with inhibiting kinase activity to induce cancer cell death i.e. see Glivec with c-Kit-. This result suggests that in the case of RET, at least, inhibiting kinase activity will probably not be sufficient to push RET-mutated tumor cell to undergo apoptosis. Thus inhibiting kinase activity should cytostatic but not cytotoxic. If so therapeutic strategy should focus on molecules affecting both the kinase activity but also the pro-apoptotic face of tyrosine kinase receptors.

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